báo cáo khoa học: "Laser ablation-based one-step generation and bio-functionalization of gold nanoparticles conjugated with aptamers" potx

Results: Using a DNA aptamer directed against streptavidin, in situ conjugation results in nanoparticles with diameters of approximately 9 nm exhibiting a high aptamer surface density 98

R E S E A R C H Open Access

Laser ablation-based one-step generation and

bio-functionalization of gold nanoparticles

conjugated with aptamers

Johanna G Walter1, Svea Petersen2, Frank Stahl1, Thomas Scheper1, Stephan Barcikowski2*

Abstract

Background: Bio-conjugated nanoparticles are important analytical tools with emerging biological and medical applications In this context, in situ conjugation of nanoparticles with biomolecules via laser ablation in an aqueous media is a highly promising one-step method for the production of functional nanoparticles resulting in highly efficient conjugation Increased yields are required, particularly considering the conjugation of cost-intensive

biomolecules like RNA aptamers

Results: Using a DNA aptamer directed against streptavidin, in situ conjugation results in nanoparticles with

diameters of approximately 9 nm exhibiting a high aptamer surface density (98 aptamers per nanoparticle) and a maximal conjugation efficiency of 40.3% We have demonstrated the functionality of the aptamer-conjugated nanoparticles using three independent analytical methods, including an agglomeration-based colorimetric assay, and solid-phase assays proving high aptamer activity To demonstrate the general applicability of the in situ

conjugation of gold nanoparticles with aptamers, we have transferred the method to an RNA aptamer directed against prostate-specific membrane antigen (PSMA) Successful detection of PSMA in human prostate cancer tissue was achieved utilizing tissue microarrays

Conclusions: In comparison to the conventional generation of bio-conjugated gold nanoparticles using chemical synthesis and subsequent bio-functionalization, the laser-ablation-based in situ conjugation is a rapid, one-step production method Due to high conjugation efficiency and productivity, in situ conjugation can be easily used for high throughput generation of gold nanoparticles conjugated with valuable biomolecules like aptamers

Background

Gold nanoparticles (AuNPs) feature unique optical

properties, including high surface plasmon resonance

(SPR), enhanced absorbance and scattering with high

quantum efficiency In addition to their resistance

against photobleaching, AuNPs perfectly fulfill

require-ments for use as colorimetric sensors and markers

For sensing or labeling of DNA targets, AuNPs can

fairly easily be functionalized with DNA via thiol

lin-kers, resulting in a highly ordered, self-assembled

monolayer (SAM) [1,2] Numerous colorimetric

appli-cations of DNA-conjugated AuNPs have already been

developed[3]

More recently, several applications of aptamer-conju-gated AuNPs have been reported[4,5] Aptamers are short, single-stranded DNA or RNA molecules that exhibit high specificity and affinity towards their corre-sponding target Thus, aptamers can be thought of as nucleic acid analogues to antibodies that can be selected

in vitro via SELEX (systematic evolution of ligands by exponential enrichment) against virtually any molecule, including proteins as well as small molecules like metal ions [6-8] Aptamer-conjugated AuNPs have already been successfully used for the detection of proteins in a dry-reagent strip biosensor, [9] for detection of throm-bin on surfaces, [4] for colorimetric detection of plate-let-derived growth factor, [5] for detection of adenosine and potassium ions in an agglomeration-based approach, [10] for detection of thrombin in a dot blot assay [11] and for targeting and therapy of cancerous cells [12,13]

* Correspondence: s.barcikowski@lzh.de

2 Laser Zentrum Hannover, Hollerithallee 8, 30419 Hannover, Germany

Full list of author information is available at the end of the article

© 2010 Walter et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in

All applications of aptamer-conjugated AuNPs

pub-lished so far have been based on chemical synthesis of

AuNPs in the presence of reducing and stabilizing

agents, and subsequent (ex situ) ligand exchange with

aptamers This ligand exchange might require heating

and buffering in order to achieve satisfactory yields and

surface coverage The latter might be limited by

interfer-ence from remaining reducing agents with the aptamer

during the replacement process Additionally, remaining

precursors and/or reducing agents might result in a

pos-sible restriction of AuNPs use in biomedical applications

[14,15]

Recently, laser ablation of gold in a liquid

environ-ment has been used for the production of AuNPs

[16,17], using surfactants for growth quenching,

result-ing in narrow nanoparticle size distributions[18] The

advantages of laser-generated AuNPs include high purity

in combination with unique surface characteristics The

Au surface of laser-generated AuNPs is partially

oxi-dized, resulting in electrostatic stabilization of the

col-loid without the need for chemical additives These

partially positively-charged AuNPs, acting as electron

acceptors, can interact directly with electron donors like

amino or thiol groups in the ablation medium[19,20]

During laser ablation, the DNA acts as a capping agent,

allowing precise size control of the resulting AuNPs, as

it has been previously reported for the addition of

cyclo-dextrines, biopolymers, etc[21] Recently, a direct

comparison of conventionalex situ conjugation of

laser-generated AuNPs and laser-ablation-basedin situ

conju-gation of AuNPs with DNA has revealed a four times

higher conjugation efficiency when using the

laser-abla-tion-based procedure In comparison to AuNPs

pro-duced by chemical synthesis and subsequent ex situ

conjugation, AuNPs generated using

laser-ablation-basedin situ conjugation exhibit up to five times higher

surface coverage[22] Hence, bio-conjugation during

laser ablation presents a rapid and efficient preparation

method, especially for the conjugation of valuable

bio-molecules like aptamers or vectors The high surface

coverage of DNA-modified AuNPs produced byin situ

conjugation may be especially advantageous for

applica-tions including cellular uptake of AuNPs In this

con-text, Giljohann et al have found that the extent of

cellular uptake of DNA-modified AuNPs can be

increased by enhancing the DNA loading[23] Moreover,

high DNA densities can also facilitate cooperative

bind-ing, resulting in increased association constants with a

given target, e.g in intracellular gene regulation[24]

Another important parameter that can be modulated via

surface coverage is the immune response induced by

DNA-modified AuNPs Higher DNA densities efficiently

limit the immune response as measured by Interferon-b

expression in mouse macrophages[25]

In spite of these benefits, the use of laser-ablation-based in situ conjugation for the generation of aptamer-conjugated AuNPs has not yet been reported

We show the functionalization of nanoparticles with aptamers during femtosecond-pulsed, laser-induced gold nanoparticle formation in an aqueous media using

a DNA aptamer directed against streptavidin as a model system In order to demonstrate the applicabil-ity of aptamer-conjugated AuNPs generated via laser ablation in complex biomedical applications, we have used an RNA aptamer directed against prostate-speci-fic membrane antigen (PSMA) for the detection of PSMA in human prostate cancer tissue utilizing tissue microarrays

Results and Discussion

Choice of aptamer orientation and spacer design

In order to ensure aptamer activity, several factors con-cerning the ability of the aptamer to fold into the cor-rect three-dimensional structure have been considered

We have previously reported the application of an apta-mer directed against streptavidin (referred to as miniS-trep) in a protein microarray format[26,27] Using this approach, we have found that the miniStrep aptamer requires an additional spacer placed between the apta-mer and the substrate to show activity that is slightly higher when immobilized via its 3’ terminus Thus, we decided to use 3’ orientation An additional oligothymi-dine (T10) spacer was placed between the disulfide group and the aptamer sequence Tymidine was chosen, since this nucleotide has the lowest affinity towards the gold surface[28] Thus, nonspecific binding of the spacer bases to gold is minimized, which should increase the surface loading and improve elevation of the aptamer away from the nanoparticle surface Taking these siderations into account, the miniStrep aptamer con-struct used in this work was the following: TCT GTG AGA CGA CGC ACC GGT CGC AGG TTT TGT CTC ACA G -T10-(CH2)3-S-S-(CH2)6OH

We decided to immobilize the anti-PSMA aptamer via the 3’terminus According to Lupold et al., the aptamer can be subjected to 3’ truncation of up to 15 nucleotides without losing its affinity to PSMA[29] Since the 3’ terminal bases are not necessary for target recognition,

we decided to omit the use of an additional oligonucleo-tide spacer Instead, hexaethylenglycol was chosen as a spacer, because it does not exhibit intermolecular repul-sion, which is one cause of low DNA loading on AuNPs [30] Furthermore, it only occupies a small surface area, which allows high packing densities,[30] and is known

to minimize nonspecific protein binding[31] Therefore, the aptamer construct used was the following: GGG AGG ACG AUG CGG AUC AGC CAU GUU UAC GUC ACU CCU UGU CAA UCC UCA UCG GCA

GAC GAC UCG CCC GA-(CH2CH2O)6-(CH2)6

-S-S-(CH2)6OH

The aptamers were directly used in the laser ablation

process without prior dithiothreitol (DTT) treatment

(Figure 1A) According to Douganet al., this does not

affect surface coverage[32] Moreover, the

mercaptohex-anol (MCH) of the mixed disulfide (aptamer-S-S-(CH2)

6OH) may serve as a co-adsorbent, eliminating

unspeci-fic binding to the gold surface, by occupying free

bind-ing sites[33] Due to the formation of a mixed

monolayer consisting of aptamers and short organic

residues, the available space for optimal aptamer folding

is enhanced (Figure 1B)

In situ conjugation

Due to rapid, one-step processing, laser-ablation-based

in situ conjugation enables fast screening of different

conjugation conditions Utilizing this high throughput

potential, we have determined optimal conjugation

con-ditions by using different concentrations of miniStrep

aptamer in a Tris(hydroxymethyl)-aminomethan (Tris)

buffer during laser ablation Per investigated

concentra-tion, the laser ablation process took less than two

min-utes A UV/VIS spectrum of AuNPs produced via laser

ablation in the presence of 5μM aptamer can be found

in Figure 2

DLS measurements demonstrate that the hydrody-namic diameter (dh) of the AuNPs increases with increasing aptamer concentrations (Figure 3A) While dh

after ablation in Tris buffer (without aptamer) is 7 nm,

dhincreases with increasing aptamer concentrations up

to 5μM, and finally reaches a plateau of approximately

60 - 70 nm (Figure 3A) We assume that this dhincrease

is a result of cumulative aptamer loading on the gold surface At low surface coverage, the aptamer lays flat

on the surface, due to non-specific binding via the lone

Figure 1 Generation of aptamer-conjugated AuNPs via in situ conjugation (A) Schematic illustration of in situ conjugation of AuNPs with aptamers during laser ablation in an aqueous aptamer solution (B) Spacer design and resulting mixed monolayer conjugated nanoparticles Mixed monolayer formation and careful spacer design contribute to correct aptamer folding.

Figure 2 UV/VIS spectrum of aptamer-conjugated AuNPs The

spectrum was obtained with an as prepared AuNP solution after in

situ conjugation with 5 μM anti-streptavidin aptamer.

Figure 3 Characterization of aptamer-conjugated AuNPs (A) Hydrodynamic diameter and Feret diameter of the aptamer AuNP conjugates as a function of the aptamer concentration used during laser ablation (B) Suggested mechanism of the observed increase of the size of the conjugates At low surface coverage, the negatively charged aptamer lays flat on the positively charged AuNP surface With increasing surface coverage, the aptamers straightened up on the surface, resulting in an increased hydrodynamic diameter Scale bars illustrate the proportions of linearized aptamer and AuNP.

nitrogen electron pairs of the nucleotides As the surface

coverage increases, the aptamers are forced to adopt a

more perpendicular conformation, due to electrostatic

repulsion of the aptamers’ negatively charged phosphate

backbones, resulting in a dh increase (Figure 3B) We

estimated the length of the aptamer (including T10

spacer) to be 21.5 nm, using a base to base distance for

ssDNA of 0.43 nm[34] For an aptamer-conjugated

AuNP of 9 nm core size, this results in a diameter of

approximately 52 nm, which is close to the observed

plateau of dh, and supports our assumption (Figure 3B)

On first sight, the AuNPs hydrodynamic diameter

increase seems to be contradictory to our previous

find-ing of a growth quenchfind-ing effect induced by increasfind-ing

DNA concentrations[19] But in contrast to our previous

work, here the ablation was performed in Tris buffer

Tris interacts with the surface of the embryonic AuNPs,

resulting in prevention of further post-ablation

nanopar-ticle agglomeration Consequently, the AuNPs produced

in Tris buffer are already stabilized by the buffer

mole-cule, resulting in reduced diameters (dh= 7.1 ± 0.8 nm

in Tris buffer versus 54.2 ± 0.6 nm in ddH2O (Figure

3A)) and a diminished influence of the oligonucleotide

concentration on the nanoparticle size This assumption

is supported by TEM analysis data The Feret diameter

(dFeret) of AuNPs produced in Tris buffer slightly

decreases from 12.1 to 7.3 nm with increasing aptamer

concentration (Figure 3A) In comparison to the Tris

molecule, the thiolated aptamer exhibits a higher affinity

towards the gold surface, resulting in better stabilization

of embryonic particles, and thus smaller AuNPs, as

detected via TEM analysis Although there may be some

portion of Tris-aptamer ligand exchange after

nanoparti-cle generation, the size quenching effect observed by

TEM analysis confirms successfulin situ

bio-conjuga-tion during laser ablabio-conjuga-tion

In addition to the AuNP size, we have determined

aptamer loading (Table 1) For AuNPs produced by

laser ablation in a 5μM aptamer solution (in situ), we

found a loading of 98 aptamers per nanoparticle,

corre-sponding to 65 pmol/cm2 This aptamer loading is

higher than the results achieved by post production (ex

situ) modification of chemically synthesized AuNPs with

short oligonucleotides (Demerset al.: 34 pmol/cm2

),[35] and the aptamer loading to chemically synthesized AuNPs reported by Huanget al (13 pmol/cm2

)[5] The high aptamer loading achieved by in situ conjugation confirms high availability of laser-generated AuNPs for bio-conjugation The conjugation efficiency was calcu-lated as the portion of provided aptamer bound to the nanoparticle surface (Table 1) At a 1.25 μM aptamer concentration, 40.3% of the available aptamer binds to the AuNPs, which demonstrates the suitability of the method for efficient conjugation of valuable biomole-cules Since we have observed no denaturation of the aptamer during laser ablation (as discussed in the next section), the remaining aptamer can be reused

After the ablation process, conjugates were slowly transferred into the aptamer selection buffer by adding NaCl and MgCl2 During this salting process, we observed precipitation of AuNPs produced at aptamer concentrations lower than 5μM The higher stability of AuNPs conjugated at aptamer concentrations of 5 μM (or higher) coincides with the plateau in the hydrody-namic diameter (Figure 3B), and indicates better stabili-zation due to higher surface coverage

All further experiments were performed with AuNPs produced in a 5 μM aptamer solution, which was a compromise between maximal aptamer density and minimal aptamer consumption (Table 1) Under these conditions, we could produce 75μg (150 μg/ml) miniS-trep-conjugated AuNPs in less than two minutes The free aptamer was removed by centrifugation In order to maintain conjugate activity, centrifugation was per-formed under rather mild conditions (16600 × g) This procedure results in a slightly increased average Feret diameter (14.6 nm) due to loss of small nanoparticles (Figure 4)

It should be noted that the high immobilization effi-ciency, and thus high aptamer consumption, results in decreasing aptamer concentrations during the laser abla-tion process As we suppose that the aptamer conjuga-tion takes place in the millisecond to second regime after the collapse of the cavitation bubble, the aptamer loading of NPs will also decrease over this period of time If more homogeneous aptamer loadings are

Table 1 Characterization of aptamer-conjugated gold nanoparticles

c (miniStrep)

a

[nm]

d Feret b

[nm]

Aptamer/AuNP Aptamer/A AuNP c

[pmol/cm 2 ]

E con d

[%]

Summary of data obtained for AuNPs conjugated with miniStrep aptamer in Tris buffer ( a

Hydrodynamic diameter, b

Feret diameter, c

Aptamers per AuNP

d

required, this can be achieved by applying higher

apta-mer concentrations and/or by shortening ablation time

Functionality of miniStrep-conjugated AuNPs

Functionality of the immobilized miniStrep aptamer was

confirmed by using three independent methods First, a

classical, agglomeration-based method was applied A

fixed amount of AuNPs (0.69 nM) conjugated with

min-iStrep aptamer was incubated with different amounts of

streptavidin (0 - 15.9 nM), and UV/VIS was detected

Since streptavidin is a tetrameric protein, agglomeration

can be observed as a red shift of SPRMax (Figure 5A)

The shift in SPRMaxincreases with increasing

concentra-tions of streptavidin, and reaches a maximum at a

strep-tavidin concentration of 2 nM In addition to the

SPRMax shift, we observed the formation of a red film

on the wall of the reaction vessel at streptavidin

concen-trations from 1 nM to 4 nM Simultaneously, we

observed a loss of AuNPs in the solution Based on

absorbance at 380 nm, the loss of particles was calcu-lated to be 86.5% We assume that the red film is com-posed of large agglomerates, while small agglomerates stay in the solution and can be detected via the shift of SPRMax and TEM analysis TEM micrographs of the agglomerates indicated a defined composition and tetra-hedral structure of these agglomerates (Figure 6) Based

on TEM analysis, we determined an agglomerate size of

35 nm (edge-to-edge length) In order to verify the pro-posed tetrahedral structure, we calculated the size of the agglomerates based on the observed shift of SPRMax, uti-lizing the“plasmon ruler equation":[36]

Δ

0 ≈0 18× 0 23

− ⎛

⎝⎜

⎞

⎠⎟

⎛

⎝

⎜

⎜

⎜

⎜

⎞

⎠

⎟

⎟

⎟

⎟

, exp

,

s D

This approximation describes the dependency between the observed shift of SPRMax (Δl) on the interparticle gap (s) and nanoparticle size (D) Using our experimen-tal results (Δl = 6 nm; l0 = 523.5 nm; D = dFeret= 14.6 nm), we calculated an interparticle distance of 9.3 nm, and an edge-to-edge length of the proposed tetrahedron

of 38.5 nm Taking into account that equation (1) is an empirical approximation established for a pair of inter-acting nanoparticles rather than for a tetramer, and con-sidering that the geometry of streptavidin is not perfectly tetrahedral, the deviation of 10% between the agglomerate sizes measured by TEM analysis and calcu-lated from the shift of SPRMaxseems to be acceptable Good agreement between the agglomeration sizes obtained using two independent methods supports the proposed tetrahedral structure of the agglomerates

At streptavidin concentrations above 2 nM, the SPRMax shift decreases, due to saturation of aptamers immobilized on the AuNPs surface with streptavidin (Figure 5B) This saturation effect is in accordance with the observations of Huanget al., who used an aptamer against a dimeric protein (platelet-derived growth fac-tor)[5]

In order to gain quantitative insight into streptavidin binding and thus aptamer activity, the AuNPs were incubated with an excess of Cy3 labeled streptavidin The agglomerates of aptamer-coated nanoparticles and attached streptavidin were removed using ultracentrifu-gation, and the amount of bound streptavidin was deter-mined by measuring the remaining streptavidin concentration in the supernatant

Since aptamer loading was determined for the whole AuNP population generated by laser ablation (dFeret= 9.0 nm), and the binding of Cy3 labeled streptavidin was performed with the AuNP subpopulation resulting from

Figure 4 TEM analysis of aptamer-conjugated AuNPs TEM

micrographs and AuNP size distributions (lognormal fit) of AuNPs

produced by laser ablation in 5 μM miniStrep solution before (A),

and after (B) removal of free aptamer using centrifugation.

removal of the free aptamer by centrifugation (dFeret = 14.6 nm), it was not possible to compare aptamer load-ing directly to the amount of bound streptavidin In order to estimate the aptamer activity, it was assumed that aptamer density (pmol/cm2) is not significantly affected by the nanoparticle diameter Based on this assumption, the aptamer loading was calculated to be 64.58 ± 1.82 pmol/cm2, and the amount of streptavidin bound to the AuNP surface was 67.23 ± 0.76 pmol/cm2, resulting in approximately 100% aptamer activity This indicates that the aptamer is not degraded during the laser ablation process, and optimal aptamer folding was achieved by careful design of the spacer

To examine the applicability of the aptamer-conju-gated AuNPs in solid phase assays, we performed a sim-ple dot blot assay (Figure 7A) Within this assay, BSA was used as a negative control BSA was chosen because

it exhibits a mildly acidic isoelectric point (pI) similar to the pI of streptavidin (pI 5-6) resulting in a comparable

Figure 5 Verification of the activity of aptamer-conjugated AuNPs (A) The shift of the SPR maximum clearly indicates the formation of agglomerates in the presence of streptavidin (B) Schematic illustration of the formation of agglomerates as a function of streptavidin

concentration: At low streptavidin concentrations, relatively small agglomerates occur (i) At medium streptavidin concentrations, the tetrameric protein induces the formation of large agglomerates (ii) An excess of streptavidin inhibits the formation of agglomerates by saturation of the aptamers bound to the nanoparticle surface (iii).

Figure 6 TEM analysis of AuNPs conjugated with aptamers

against streptavidin TEM micrographs of AuNPs without

streptavidin (i), and after incubation with streptavidin (ii) The insert

displays a scheme of the proposed composition of the

agglomerates Please note that the agglomerates are displayed in a

simplified manner in the scheme, de facto streptavidin is not planar

but tetrahedral.

negative net charge of the two proteins under the given

conditions (pH 7.4) In order to exclude the possibility

of electrostatic interactions between the negatively

charged proteins and the positively charged AuNPs, the

experiment was repeated with unconjugated

nanoparti-cles The nanoparticle aptamer conjugates bind only to

the immobilized streptavidin, and no binding can be

observed to BSA Using unconjugated nanoparticles, no

binding of AuNPs to the immobilized proteins occurred

(data not shown) This clearly demonstrates the specific

binding of AuNPs to streptavidin via the aptamer

conju-gated to the nanoparticle surface

The AuNPs bound to streptavidin during the dot blot

assay were further analyzed via ESEM The ESEM

micrograph affirms the Feret diameter of the

nanoparti-cles determined by TEM (Figure 7B)

Functionality of anti-PSMA-conjugated AuNPs

Encouraged by the positive performance of the dot blot

assay, our next aim was to prove the applicability of

aptamer-conjugated AuNPs in more complex and

demanding solid-phase assays Therefore, we used

AuNPs conjugated with an aptamer directed against

PSMA for detection of PSMA in prostate cancer

(adeno-carcinoma) tissue sections

AuNPs conjugated with anti-PSMA aptamer show a

staining pattern similar to anti-PSMA antibody In both

cases, a positive staining of acinar epithelial cells was

observed (Figure 8) In tissue sections treated with

anti-PSMA aptamer-conjugated AuNPs, an additional staining

of muscle cells was observed that was not detected in the

positive control To ensure that the binding to PSMA is

based on the affinity of the anti-PSMA aptamer rather

than on electrostatic interaction between the target

pro-tein and the highly negatively charged aptamers, AuNPs

conjugated with anti-streptavidin aptamers were used

Since the negative control does not show positive binding

to epithelial cells or false positive binding to muscle cells,

we assume the binding of anti-PSMA conjugates to mus-cle cells to be induced by the specific three-dimensional structure of the anti-PSMA aptamer A positive staining

of smooth muscle cells in prostate cancer has also been reported for one monoclonal PSMA antibody (7E11),[37] and some authors assume that there may be a “PSMA-like” target in smooth muscle cells[38,39] Following this consideration, the binding of AuNPs conjugated with anti-PSMA aptamer to muscle cells may be the result of cross-reactivity of the aptamer with this unknown

“PSMA-like” target In summary, our results demonstrate that the anti-PSMA aptamer AuNP conjugates can detect PSMA in acinar epithelial cells of human prostate cancer This exemplifies the broad applicability of

aptamer-Figure 7 Dot blot Dot blot detection of streptavidin, using

aptamer-conjugated gold nanoparticles (A) ESEM image of

nanoparticles bound to streptavidin immobilized on the

nitrocellulose membrane (B).

Figure 8 Detection of PSMA in human prostate cancer tissue Detection of PSMA positive structures in prostate cancer tissue sections by immunohistochemical staining using anti-PSMA aptamer (PSMA apt)-conjugated AuNPs As a negative control, AuNPs conjugated with miniStrep aptamer (miniStrep apt) were used A polyclonal antibody directed against PSMA (PSMA pAb) was used as

a positive control Positive control was additionally stained with Haematoxylin and Eosin Black arrows indicate specific staining, while white arrows flag unspecific binding.

conjugated AuNPs, even in highly complex biological

matrices and bio-imaging applications

Conclusions

We have demonstrated the suitability of

laser-ablation-basedin situ bio-conjugation for the production of

func-tional, aptamer-conjugated gold nanoparticles Exploiting

the potential of this rapid, one-step method for high

throughput screening, we have optimized the conjugation

regarding aptamer loading and conjugation efficiency To

address the general applicability of the method, we have

utilized two different aptamers composed of DNA and

RNA The high degree of aptamer activity determined on

AuNP surface verifies that there is no heat-induced

dena-turation of the aptamer during laser ablation We have

proven the functionality of conjugates using three

differ-ent methods (agglomeration-based assay, dot blot assay,

tissue microarray), indicating the broad applicability of

aptamer-conjugated gold nanoparticles for bio-analytical

applications, even in highly demanding assays Moreover,

in situ conjugation avoids possible contamination by

toxic educts, residual reducing agents or preservatives

Thus, this method could also be especially advantageous

for use in medical applications

Sincein situ conjugation is a fast and simple one-step

approach to generate pure conjugated AuNPs with high

conjugation efficiency and productivity, it can be easily

used for the high throughput production of large

amounts of different conjugated nanoparticles The

higher conjugation efficiencies are beneficial for

high-priced biomolecules, and the comparably high surface

coverage is desirable for cellular uptake, which depends

on the DNA density on the AuNPs surface Moreover,

such high surface densities may assist cooperative

bind-ing and may decrease the immune response against

AuNPs

Methods

Materials

All chemicals were purchased from Sigma-Aldrich

(Steinheim, Germany) or Fluka Chemie AG

(Tauf-kirchen, Germany), and used as received The aptamer

against streptavidin (TCT GTG AGA CGA CGC ACC

GGT CGC AGG TTT TGT CTC ACA G -T10-(CH2)3

-S-S-(CH2)6OH, referred to as miniStrep) [26] and

anti-PSMA aptamer (GGG AGG ACG AUG CGG AUC

AGC CAU GUU UAC GUC ACU CCU UGU CAA

UCC UCA UCG GCA GAC GAC UCG CCC

GA-(CH2CH2O)6-(CH2)6-S-S-(CH2)6OH) [29] were

pur-chased from Biospring GmbH (Frankfurt, Germany)

The gold foil was 0.1 mm thick and had >99.99% purity,

and was obtained from Goodfellow GmbH (Bad

Nau-heim, Germany)

Generation of aptamer-conjugated AuNPs

Laser ablation was performed in the same buffer system the aptamer was originally selected in For the miniStrep aptamer, 50 mM Tris(hydroxymethyl)-aminomethan (Tris) pH 8.0 was used, and the anti-PSMA aptamer was conjugated in 20 mM N’-2-Hydroxyethylpiperazine-N’-2 ethanesulphonic acid (HEPES) pH 7.4 Laser generation

of AuNPs was performed utilizing a Spitfire Pro femto-second laser system (Spectra-Physics) providing 120 fs laser pulses at a wavelength of 800 nm 5×5 mm gold foils were placed in the wells of a 24 well plate filled with 500μl of aptamer solution in the respective buffer Ablation was performed while moving the plate at a constant speed of 60 mm×min-1 in a spiral (outer radius: 3 mm, inner radius: 1.5 mm), using an axis sys-tem Recently, we have optimized the laser parameters for laser-ablation-based generation of DNA-conjugated AuNPs[19] Here, laser fluence was optimized in regard

to maximal productivity, while avoiding degradation of the oligonucleotide In the present study, the optimized parameters were chosen, the pulse energy was fixed at

100μJ, and the repetition rate was 5 kHz In order to avoid heat-induced degradation of the aptamer, the focus position was adjusted to be 2 mm beneath the focus position determined in air[19]

Post-generation processing of the aptamer-conjugated AuNPs

After laser ablation, the conjugates were allowed to age overnight at 4°C before NaCl was added in increments

of 25 mM by addition of 2 M NaCl in Tris-Cl or HEPES respectively After each NaCl addition, the col-loidal solution was mixed and incubated for 1 h at room temperature The addition of MgCl2 and CaCl2was per-formed after another overnight incubation at 4°C, by addition of 1 M MgCl2 and 1 M CaCl2 Final buffer compositions were the following: miniStrep: 150 mM NaCl, 10 mM MgCl2, 50 mM Tris-Cl pH 8.0; anti-PSMA: 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.05% Tween 20, 20 mM HEPES pH 7.4

To remove the free aptamer, the ablation medium was centrifuged for 15 min at 15000 rpm The supernatant was transferred into a new centrifugal tube and centri-fuged for another 30 min The supernatant was dis-carded, and the pellets were pooled and resuspended in the respective buffer This process was repeated 4 times

Characterization methods

UV/VIS spectra of the AuNP solutions were recorded using a Shimadzu 1650 spectrophotometer In order to determine the AuNP concentration, the absorption at

380 nm (mainly corresponding to the interband transi-tion of gold) was measured Intensities were converted

to AuNP mass concentrations by interpolation from a

linear standard calibration curve (R2 = 0.99) Standard

curves were prepared with known concentrations of

AuNP produced by weighing a gold target three times

before and after ablation

Transmission electron micrographs (TEM) were

com-missioned at Stiftung Tierärztliche Hochschule, Institut

für Pathologie (Prof Dr W Baumgärtner, Kerstin

Rohn), and were obtained by utilizing a TEM Philip

CM30 with a 0.23 nm resolution One drop of the

col-loidal solution was placed on a carbon-coated,

formvar-covered copper grid, and then dried at room

tempera-ture Given diameters were averaged for at least 200

AuNPs Dynamic light scattering (DLS) measurements

were performed, using a Zetasizer ZS (Malvern) Three

consecutive measurements were carried out and average

values are presented

The amount of aptamer bound per nanoparticle was

determined by measuring the concentration of the

unbound aptamer Aptamer-conjugated AuNPs were

removed by ultracentrifugation (Beckman Coulter

Optima Max, 30000 × g), and the adsorption of the

supernatant was measured at 260 nm against a serial

dilution of aptamer in Tris buffer Mean values of three

measurements are presented

Determination of miniStrep aptamer functionality

The agglomeration-based streptavidin assay was

per-formed by incubating a fixed amount of

miniStrep-con-jugated AuNPs (0.69 nM) with varying concentrations

of streptavidin (0 - 15.9 nM) for 16 h at room

tempera-ture UV/VIS was measured to monitor the shift of

SPRMax

Furthermore, the aptamer activity was determined in a

“golden blot”[40] format similar to the method

pub-lished by Wanget al[11] In brief, streptavidin (0.5 μl, 1

mg/ml in PBS) was spotted in 10 replicates onto a

nitro-cellulose membrane (Sartorius, Goettingen, Germany)

After 1 h incubation at room temperature, blocking of

the membrane was performed with 1% BSA in

miniS-trep selection buffer The membrane was washed in the

same buffer and incubated with a solution of AuNPs

(20 μg/ml) for 2 h Finally, the membrane was washed

with miniStrep selection buffer As a negative control,

BSA (0.5μl, 1 mg/ml in PBS) was spotted on the

mem-brane Furthermore, the experiment was repeated with

“bare” AuNP produced in Tris buffer in the absence of

aptamer In this experiment, the miniStrep selection

buffer was replaced by 50 mM Tris-Cl pH 8.0, in order

to maintain colloidal stability of the non-stabilized

nano-particles Environmental scanning electron microscopy

(ESEM) of the membrane after incubation with AuNPs

was performed with a Quanta 400 F (FEI, Eindhoven,

Netherlands) in low vacuum conditions A piece of

membrane was placed on an aluminum holder and visualized without previous sputtering

In order to determine the activity of the miniStrep aptamer bound to the AuNP surface, the conjugate (28.5 μg/ml, 0.15 nM) was incubated with Cy3-labeled streptavidin (166.7 μg/ml, 2.8 μM) for 16 h at room temperature, in the dark The conjugates and bound streptavidin were removed by ultracentrifugation The amount of streptavidin bound to the nanoparticles was determined by measuring the streptavidin concentration remaining in the supernatant, utilizing a Fluoroskan ascent fluorescence plate reader (Ex: 544 nm, Em: 590 nm) Mean values of 4 measurements are presented

Determination of anti-PSMA aptamer functionality

The activity of anti-PSMA aptamers conjugated to AuNPs was investigated, using a tissue microarray con-sisting of paraffin-embedded prostate cancer tissues (US Biomax, Rockville, MD, USA) After baking the slides at 60°C for 30 min, paraffin was removed using two wash-ing steps in xylene (10 min each) The tissue arrays were rehydrated by consecutive washes in 100%, 95% and 70% ethanol, followed by a washing step in ddH2O (5 min each) Antigen retrieval was performed by pla-cing the slides in 0.01 M sodium citrate pH 6.0 for

15 min at 95°C Consequently slides were washed with anti-PSMA aptamer selection buffer, and blocked in 5% goat serum (Millipore) in the same buffer The anti-PSMA selection buffer was used for all consequent assay steps Incubation with the aptamer-modified AuNPs (20μg/ml) was performed for 2 h at 20°C and 300 rpm

in an Eppendorf shaker equipped with a slide adaptor, after placing a secure seal incubation chamber (Grace Biolabs, Bend, OR, USA) filled with 800 μl of the respective AuNP solution on the slide Slides were washed two times for 5 min with 1% goat serum, and fixed for 15 min with 2.5% glutaraldehyde solution Sil-ver enhancement was performed using a silSil-ver enhancer kit (Sigma), according to the instructions provided by the manufacturer

AuNPs conjugated with miniStrep Aptamer in HEPES buffer were chosen as a negative control All washing and incubation steps were performed as described above As

a positive control, a rabbit anti-PSMA antibody directed against the C-terminal domain of human PSMA (Milli-pore) was used[41] Here, all washing and incubation steps were performed using PBS After incubation with 2.5 μg/ml rabbit anti-PSMA for 2 h, the slides were washed two times for 5 min with 1% goat serum, and consequently incubated with a 1:20 dilution of 12 nm colloidal gold conjugated with goat anti-rabbit IgG (Jack-son Immuno Research; OD at 520 nm of stock solution: 2) for 1.5 h High background of developed tissue arrays was removed as described by Springallet al[42]

This work was funded by the German Research Foundation Society DFG

within the Excellence Cluster REBIRTH (From Regenerative Biology to

Reconstructive Therapy) The authors thank Prof C Urbanke, PD U Curth

and Frank Hartmann (Medizinische Hochschule Hannover) for the possibility

to use the ultracentrifugation facilities.

Author details

1 Institut für Technische Chemie, Leibniz Universität Hannover, Callinstrasse 3,

30167 Hannover, Germany.2Laser Zentrum Hannover, Hollerithallee 8, 30419

Hannover, Germany.

Authors ’ contributions

JGW and SP carried out the in situ conjugations and partial drafting of the

manuscript JGW carried out the determination of aptamer functionality.

JGW and FS carried out the tissue microarray experiments SB carried out

the principal study design, manuscript drafting and supervision of

nanoparticle generation TS participated in the conception design and

supervised aptamer-related work All authors read and approved the final

manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 30 March 2010 Accepted: 23 August 2010

Published: 23 August 2010

References

1 Alivisatos AP, Johnsson KP, Peng X, Wilson TE, Loweth CJ, Bruchez MP Jr,

Schultz PG: Organization of ‘nanocrystal molecules’ using DNA Nature

1996, 382:609-611.

2 Mirkin CA, Letsinger RL, Mucic RC, Storhoff JJ: A DNA-based method for

rationally assembling nanoparticles into macroscopic materials Nature

1996, 382:607-609.

3 Sato K, Hosokawa K, Maeda M: Colorimetric biosensors based on

DNA-nanoparticle conjugates Anal Sci 2007, 23:17-20.

4 Pavlov V, Xiao Y, Shlyahovsky B, Willner I: Aptamer-functionalized Au

nanoparticles for the amplified optical detection of thrombin J Am

Chem Soc 2004, 126:11768-11769.

5 Huang CC, Huang YF, Cao Z, Tan W, Chang HT: Aptamer-modified gold

nanoparticles for colorimetric determination of platelet-derived growth

factors and their receptors Anal Chem 2005, 77:5735-5741.

6 Tuerk C, Gold L: Systematic evolution of ligands by exponential

enrichment: RNA ligands to bacteriophage T4 DNA polymerase Science

1990, 249:505-510.

7 Ellington AD, Szostak JW: In vitro selection of RNA molecules that bind

specific ligands Nature 1990, 346:818-822.

8 Robertson DL, Joyce GF: Selection in vitro of an RNA enzyme that

specifically cleaves single-stranded DNA Nature 1990, 344:467-468.

9 Xu H, Mao X, Zeng Q, Wang S, Kawde AN, Liu G: Aptamer-functionalized

gold nanoparticles as probes in a dry-reagent strip biosensor for protein

analysis Anal Chem 2009, 81:669-675.

10 Zhao W, Chiuman W, Lam JC, McManus SA, Chen W, Cui Y, Pelton R,

Brook MA, Li Y: DNA aptamer folding on gold nanoparticles: from colloid

chemistry to biosensors J Am Chem Soc 2008, 130:3610-3618.

11 Wang Y, Li D, Ren W, Liu Z, Dong S, Wang E: Ultrasensitive colorimetric

detection of protein by aptamer-Au nanoparticles conjugates based on

a dot-blot assay Chem Commun (Camb) 2008, 2520-2522.

12 Medley CD, Smith JE, Tang Z, Wu Y, Bamrungsap S, Tan W: Gold

nanoparticle-based colorimetric assay for the direct detection of

cancerous cells Anal Chem 2008, 80:1067-1072.

13 Huang YF, Sefah K, Bamrungsap S, Chang HT, Tan W: Selective

photothermal therapy for mixed cancer cells using aptamer-conjugated

nanorods Langmuir 2008, 24:11860-11865.

14 Besner S, Kabashin A, Winnik F, Meunier M: Ultrafast laser based “green”

synthesis of non-toxic nanoparticles in aqueous solutions Applied Physics

A 2008, 93:955-959.

15 Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD: Gold nanoparticles

are taken up by human cells but do not cause acute cytotoxicity Small

2005, 1:325-327.

16 Sylvestre J, Kabashin A, Sacher E, Meunier M: Femtosecond laser ablation

of gold in water: influence of the laser-produced plasma on the nanoparticle size distribution Applied Physics A 2005, 80:753-758.

17 Barcikowski S, Hahn A, Kabashin A, Chichkov B: Properties of nanoparticles generated during femtosecond laser machining in air and water Applied Physics A 2007, 87:47-55.

18 Mafune F, Kohno J, Takeda Y, Kondow T, Sawabe H: Formation of gold nanoparticles by laser ablation in aqueous solution of surfactant J PHYS CHEM B 2001, 105:5114-5120.

19 Petersen S, Barcikowski S: In Situ Bioconjugation: Single Step Approach to Tailored Nanoparticle-Bioconjugates by Ultrashort Pulsed Laser Ablation ADV FUNCT MATER 2009, 19:1167-1172.

20 Petersen S, Jakobi J, Barcikowski S: In situ bioconjugation-Novel laser based approach to pure nanoparticle-conjugates APPL SURF SCI 2009, 255:5435-5438.

21 Sylvestre JP, Kabashin AV, Sacher E, Meunier M, Luong JH: Stabilization and size control of gold nanoparticles during laser ablation in aqueous cyclodextrins J Am Chem Soc 2004, 126:7176-7177.

22 Petersen S, Barcikowski S: Conjugation Efficiency of Laser-Based Bioconjugation of Gold Nanoparticles with Nucleic Acids J Phys Chem C

2009, 113:19830-19835.

23 Giljohann DA, Seferos DS, Patel PC, Millstone JE, Rosi NL, Mirkin CA: Oligonucleotide loading determines cellular uptake of DNA-modified gold nanoparticles Nano Lett 2007, 7:3818-3821.

24 Rosi NL, Giljohann DA, Thaxton CS, Lytton-Jean AK, Han MS, Mirkin CA: Oligonucleotide-modified gold nanoparticles for intracellular gene regulation Science 2006, 312:1027-1030.

25 Massich MD, Giljohann DA, Seferos DS, Ludlow LE, Horvath CM, Mirkin CA: Regulating immune response using polyvalent nucleic acid-gold nanoparticle conjugates Mol Pharm 2009, 6:1934-1940.

26 Bittker JA, Le BV, Liu DR: Nucleic acid evolution and minimization by nonhomologous random recombination Nat Biotechnol 2002, 20:1024-1029.

27 Walter JG, Kokpinar O, Friehs K, Stahl F, Scheper T: Systematic investigation

of optimal aptamer immobilization for protein-microarray applications Anal Chem 2008, 80:7372-7378.

28 Kimura-Suda H, Petrovykh DY, Tarlov MJ, Whitman LJ: Base-dependent competitive adsorption of single-stranded DNA on gold J Am Chem Soc

2003, 125:9014-9015.

29 Lupold SE, Hicke BJ, Lin Y, Coffey DS: Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen Cancer Res 2002, 62:4029-4033.

30 Hurst SJ, Lytton-Jean AK, Mirkin CA: Maximizing DNA loading on a range

of gold nanoparticle sizes Anal Chem 2006, 78:8313-8318.

31 Zhang F, Skoda MW, Jacobs RM, Zorn S, Martin RA, Martin CM, Clark GF, Goerigk G, Schreiber F: Gold nanoparticles decorated with oligo(ethylene glycol) thiols: protein resistance and colloidal stability J Phys Chem A

2007, 111:12229-12237.

32 Dougan JA, Karlsson C, Smith WE, Graham D: Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides Nucleic Acids Res 2007, 35:3668-3675.

33 Balamurugan S, Obubuafo A, Soper SA, Spivak DA: Surface immobilization methods for aptamer diagnostic applications Anal Bioanal Chem 2008, 390:1009-1021.

34 Park S, Brown K, Hamad-Schifferli K: Changes in oligonucleotide conformation on nanoparticle surfaces by modification with mercaptohexanol NANO LETT 2004, 4:1925-1929.

35 Demers LM, Mirkin CA, Mucic RC, Reynolds RA, Letsinger RL, Elghanian R, Viswanadham G: A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles Anal Chem

2000, 72:5535-5541.

36 Jain P, Huang W, El-Sayed M: On the universal scaling behavior of the distance decay of plasmon coupling in metal nanoparticle pairs: A plasmon ruler equation NANO LETT 2007, 7:2080-2088.

37 Kinoshita Y, Kuratsukuri K, Landas S, Imaida K, Rovito PM Jr, Wang CY, Haas GP: Expression of prostate-specific membrane antigen in normal and malignant human tissues World J Surg 2006, 30:628-636.

38 Chang SS, Reuter VE, Heston WD, Bander NH, Grauer LS, Gaudin PB: Five different anti-prostate-specific membrane antigen (PSMA) antibodies